1. Field of the Invention
The present invention relates to a method for obtaining components by fracturing the cell membrane of leucocytes in human blood, and separating and collecting the leucocyte components, individually.
2. Prior Art
Conventionally, it has been commonly known that erythrocytes of human blood mainly act as carriers for carrying a large amount of oxygen and carbon dioxide at a high speed, and leucocytes act as phagocytes against various bacteria. However, in practical scenes, erythrocytes also crowd at the inflamed part where a great deal of bacteria exist. It seems that thus crowded erythrocytes act as any biological function in the inflamed part. In addition to this phenomenon, the biological correlation between erythrocytes and leucocytes in such inflamed and bacterial affected parts has not been clearly known. The inventor of the present application previously submitted the patent application titled xe2x80x9cMethod for fractionating red blood cellsxe2x80x9d Japanese Patent Application No. Tokugan-Hei 8-215552/1996, corresponding to European Patent Application No. 97102952.5 titled xe2x80x9cMethod for fractionating red blood cells and antibacterial materials or bacterial proliferation inhibitors produced therebyxe2x80x9d. The inventor observed the biological function between the erythrocytes and bacteria included in respective three layers fractionated in accordance with the previously applied method.
In detail, the three erythrocyte-samples from the fractionated three layers; i.e., top, middle, and bottom layers, were respectively added into liquid culture medium, and then bacteria sample was inoculated into all of the culture medium to observe the function of live erythrocytes against the inoculated bacteria. In the top layer the bacteria were surrounded with the erythrocytes and thus their bacterial proliferation were inhibited. Although the top layer included leucocytes, these leucocytes did not provide any phagocytosis to the bacteria. The erythrocytes included in the middle layer did not show any aggressive motions towards the bacteria, but the bacterial proliferation were inhibited. The middle layer also included a small amount of leucocytes, which did not show any motions towards the bacteria. The erythrocytes included in the bottom layer did not show any aggressive motions towards the bacteria. Thus, the bacterial proliferation were observed. The bottom layer did not include leucocytes at all.
Since the number of leucocytes in human blood are remarkably increased when bacterial inflammation causes or the size of specific leucocyte is extremely enlarged in bacterial inflamed part, it has been conventionally realized that these phenomena are resulted from the phagocytosis of leucocytes. However, as disclosed in the previously applied invention xe2x80x9cMethod for fractionating red blood cellsxe2x80x9d the observation on the action and change of live erythrocytes and leucocytes around bacteria proves that conventional knowledge; i.e., leucocytes only attack bacteria, is not correct.
According to conventional knowledge, erythrocytes comprise single type blood cells having a uniform figure and the same characteristics, while leucocytes comprise several different real cells having nucleus and cytoplasm. Conventionally, leucocytes are classified depending on appearance and staining property into five groups, neutrophil leucocyte, eosinophile leucocyte, basophilic leucocyte, lymphoid cell, and monocyte as disclosed in xe2x80x9cSeikagaku Jiten (Encyclopedia of Biochemistry)xe2x80x9d published by Tokyo Kagakudoj in. Conventional method for laboratory tests of erythrocytes and leucocytes have been performed as following steps. One drop of blood is placed on a glass plate and is spread into a thin layer using the end of another glass plate. After drying, this blood sample is stained with various staining agents and then the stained sample is observed through an optical microscope. This conventional method, however, leads to following three defects.
(1) As the blood sample is dried, the cells are mummified. Therefore this conventional method is too primitive and coarse to observe such delicate, soft, and volatile cells as the erythrocytes and leucocytes.
(2) In the staining technique, the blood cells and components difficult of staining cannot be observed at all. The part that does not stain easily is ignored and the part that stains easily is over-stained darkly. Accordingly, minute changes cannot be observed.
(3) In biological researches, a phenomenon should be observed from the beginning to the end covering as long a period as possible. The biological truth cannot be known unless the state changing with time is grasped.
Conventionally, Ficoll-Conray method has been known to separate and collect lymphoid cells from leucocytes. Thus collected lymphoid cells have been used in various manners to study the immune system about the leucocytes in human blood. For example, the inventor of the present application found out that remarkable effects were not resulted when the lymphoid cells obtained from a healthy person were administered to the rheumatics, but remarkable effects were resulted when cultured leucocytes of a healthy person were administered to the same patient. Further, the inventor has continuously researched on clinical cases of many rheumatics administered with the cultured leucocytes, and confirmed the superior effects in medical treatment for the rheumatics. The inventor reported and published such therapeutic effects at many academic meetings and in bulletins; as an example, International Rheumatism Conference 1981 held in Paris.
It has been assumed that stale cells are broken into water-soluble fine pieces and transported by flowing blood towards liver and kidney. Although the liver and kidney act as the final wastes treatment, the processes of decomposition and water-solubilization of stale cells prior to the final wastes treatment are not clarified. The inventor used the bottom layer (excluding leucocytes) of the fractionated three layers provided by xe2x80x9cMethod for fractionating red blood cellsxe2x80x9d to observe the wastes treatment function of leucocytes. In detail, the bottom layer was added with frozen leucocytes or live leucocytes of the same blood type, and then cultured. As a result, this cultured bottom layer showed that the blood cells of the bottom layer were changed to minute and water-soluble particles rather than the non-treated sample free from leucocytes. Additionally, this effect appeared remarkably in the case of using the frozen leucocytes rather than the live leucocytes. This phenomenon means following two functions. First, leucocytes have the function for treating stale or perished erythrocytes as wastes. The second function is anticipated from the phenomenon of the frozen leucocytes having superior effect to live leucocytes. Some components spread out of the leucocyte-cells fractured during freezing step may act as agents which accelerate the decomposition and water-solubilization of stale or perished erythrocytes.
As discussed above, many clinical tests proved that the cultured leucocytes obtained from a healthy person had effect on specific diseases such as rheumatic disease. However, in actual therapeutic scenes, the cultured leucocytes should be subjected to various tests, for example, the blood check for AIDS virus, prior to administration, and further, should be prepared and stored in a specially controlled space such as an aseptic culture room with meticulous care. Thus therapeutic cost will become high. As an example in an actually performed test-therapy, an administration of cultured leucocytes is performed per four weeks; one course includes six times of administrations; and at least four courses are desired. One administration needs about Yen 50,000 (US$ 550), and thus totally four courses need about Yen 1,200,000 (US$ 13,200). This cost is extremely high for ordinary patients, and therefore on the present stage, actual therapies use steroid drugs which produce harmful aftereffects and anti-inflammatory or analgesic agents which produce temporary pain-free effect.
According to the above observations, the inventor concludes that the correlation between the leucocyte components and the erythrocytes, the therapeutic function of the leucocyte components for various bacteria, and the function of the leucocyte components for treating stale or perished cells should be clarified in order to produce a new drug which is free from harmful aftereffects produced by antibiotics and steroid drugs. Further such leucocytes induced drugs are expected to be applied to the therapy for various cancers, hepatic disease, and renal disease at a low cost. If hepatic disease and renal disease are healed or improved by such new drugs, it is expected to decrease lever and kidney transplant operations which are risky, complicated and high cost operations.
It is therefore a primary object of the present invention to provide a method for fracturing the cell membrane of leucocytes in human blood to separate and collect leucocyte components.
Another object of the present invention is to provide a method to separate and collect the leucocyte components under the near-live condition.
In order to accomplish the above objects, the present invention provides a method comprising (A) a first step for fracturing the cell membrane of leucocytes in human blood by physical means, and (B) a second step for separating and collecting the leucocyte components from the blood liquid resulted from the first step, containing the leucocytes with fractured cell membranes, by means of a centrifugal precipitation technique or an electrophoresis technique.
Thus separated and collected leucocyte components may be respectively subjected to various therapeutic tests using blood samples collected from patients suffering from various diseases to know the therapeutic effects. One typical example of these therapeutic tests is performed in the following steps.
(1) 5 to 10 ml of blood sample is taken from a patient suffering from cancer, hepatic disease, or renal disease and then separated into three layers; top layer, middle layer, and bottom layer, in the same manner as shown in the method for fractionating red blood cells, disclosed in the prior invention. These three layers are cultured respectively, and their changes are observed through a phase-contrast microscope for a predetermined period. These observed data are recorded by a still camera, video camera, or the like, and used as reference for judging the therapeutic effects.
(2) The three layers are also obtained from the same patient""s blood in the same fractionating method, and then added with one group of the leucocyte components separated from a healthy person""s blood. The three layers are cultured and the changes of erythrocytes are observed through the same phase-contrast microscope at a predetermined time interval. According to the above works (1) and (2), it is possible to judge which leucocyte component has the therapeutic effect on which kind of diseases.
The physical means used in the first step (A) for fracturing the cell membranes of leucocytes may be selected from (a) a supersonic method for applying the supersonic of 1 MHz to 50 MHz to the blood liquid containing leucocytes to fracture the cell membranes of leucocytes by the vibration caused by the supersonic; (b) a laser method for irradiating the laser of 10 to 100 mW, 50/cm2 for several seconds to several minutes (about 3 minutes) to the same point in the blood liquid containing leucocytes to fracture the cell membranes; (c) an osmotic pressure method for changing the osmotic pressure of the blood liquid containing leucocytes to fracture the cell membranes; (d) a freezing and defrosting method for freezing the blood liquid containing leucocytes at the temperature range from xe2x88x925 degrees to the absolute zero point and then defrosting this frozen liquid at a room temperature (about 20 degrees) to fracture the cell membranes; and (e) a vacuum method for rapid-reducing the pressure in a vacuum chamber to fracture the cell membranes of the blood liquid containing leucocytes set in the chamber.
The second step (B) for separating and collecting the leucocyte component includes a centrifugal precipitation technique which stirs the blood liquid containing the leucocytes with cell membranes fractured by the first step (A), and then separates the stirred liquid into multiple layers corresponding the leucocyte components by the centrifugal precipitation work. Alternatively, the second step (B) includes an electrophoresis technique which separates the blood liquid containing the leucocytes with fractured cell membranes by the first step (A) into multiple parts corresponding the leucocyte components by the electrophoresis work. Finally, the separated layers or parts are collected separately.
The first step (A) may use the cultured leucocytes obtained from the specific persons who are judged healthy through predetermined health checks.
These and other objects and many of the attendant advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings.